(Received 28 May 1958) According to Richter (1957) the ability of rats to make dietary selections

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1 138 J. Physiol. (1958) I44, I38-I47 THE EFFECTS OF METHYLPENTYNOL ON ETHANOL DRINKING AND ON WATER METABOLISM IN RATS BY S. E. DICKER From the Department of Pharmacology, University College London (Received 28 May 1958) According to Richter (1957) the ability of rats to make dietary selections depends on the sense of taste more than on the effects produced by substances after ingestion. As methylpentynol apparently dulls the sense of discrimination of rats (Halpern & Lehmann, 1957; Watson, 1958) it was thought of interest to see whether the administration of the drug to rats might change the distaste which they usually have for comparatively concentrated solutions of ethanol and induce them to drink solutions which they normally refuse. This in turn has led to study of the effect of methylpentynol on the water metabolism of rats. A preliminary account of this investigation has been communicated to the Physiological Society (Dicker, 1958). METHODS Rats of similar weight ( g) and age (5-6 months) were kept either singly or in pairs in metabolism cages at 210 C. Food and fluid intake, urine and faeces excretion were measured and the animals were weighed daily. The diet was the same as that described previously (Dicker & Nunn, 1957; its calorie yield was 400 cal/100 g. The food total water content (metabolic water + moisture) was 65 ml./100 g. The average water content of faeces was 65 ml./100 g. Extrarenal water loss was estimated as the difference between total water intake ( = water drunk + metabolic water + food moisture) and total water excretion ( = urine + water content of faeces). Water was offered from two similar bottles, one of which might alternatively contain an ethanol solution. To avoid the effects of habituation the position of the bottles in the cages was interchanged every day (Gillespie & Lucas, 1958). The release of pressor or oxytocic activity from the neurohypophysis was investigated, using rats of either sex anaesthetized with urethane (0-7 ml./100 g of a 25% urethane solution injected subeutaneously). The blood pressure was recorded with a mercury manometer from the femoral artery of male rats injected with 1.0 mg dibenamine (Dekanski, 1952). Uterine contractions were recorded in situ using a Cushny (1897) myocardiograph fixed on the horn of the uterus of rats given stilboestrol (0.01 mg/100 g) by intramuscular injection on the previous day. In both preparations one common carotid artery and a tail vein were cannulated for injections. Drugs: for injection a solution of 3-methyl-pentyne-ol-3 (=methylpentynol) and for oral administration a solution of methylpentynol carbamate dissolved in tepid water were administered. Pitressin (Parke, Davis and Co.), batch LT488H, and Pitocin (Parke, Davis and Co.), batch LU887K, were used as vasopressin and oxytocin solutions respectively.

2 METHYLPENTYNOL IN RATS 139 RESULTS The present experiments were carried out on twenty-four rats during the winter. This may be the reason why the food ( g/100 g/24 hr) and the water ( ml./100 g/24 hr) intake were smaller than that reported for rats observed over a period of a year (Dicker & Nunn, 1957). Intake by rats of ethanol solutions of various concentrations When a solution of 4% (v/v) ethanol was substituted for water in one of the two drinking bottles (Fig. 1, days 6-8), rats showed a marked preference for ethanol, which they drank to the practical exclusion of water (Richter, 1957). Total fluid intake was increased and there was a rise in urine excretion I Days Fig. 1. Water, ethanol and food consumption in rats. Q-, Water drunk (ml./100 g/24 hr); 0-0, food eaten (g/100 g/24 hr); x - x, urine excretion (ml./100 g/24 hr); *_-, ethanol solutions drunk (ml./100 g/24 hr). The numerals above the arrows indicate the concentration of the ethanol solution offered on that and following days. The results are averages obtained from observations on three pairs of rats.

3 140 S. E. DICKER (Eggleton, 1942; van Dyke & Ames, 1951) from (6) to (6) ml./100 g/24 hr (Fig. 1). The total calorie intake was /100 g/24 hr; it remained unaffected by the drinking of ethanol, the animals reducing their food consumption in proportion to the intake of calories from ethanol. The calories from ethanol amounted to 4f /100 g/24 hr. There was no appreciable change in the general metabolism when the concentration of ethanol was increased by 1 % (v/v) at a time until it reached 9 % (v/v). Above that concentration the volume of ethanol solution drunk decreased (Fig. 1), though the mean calorie intake from ethanol remained unchanged at cal/100 g/24 hr. The deficit of liquid resulting from the decreased consumption of ethanol solutions was made good by an increase of water intake. Preference for ethanol disappeared if its concentration exceeded 15% (v/v) (Fig. 1). Rats drank, for example, 7-0 ml./100 g/24 hr of a 12 % (v/v) ethanol solution (Fig. 1 days 24-26) without any noticeable signs of intoxication. This is in contrast with the effect of a single oral dose of 5 0 ml./100 g of a 12% (v/v) ethanol solution which produces a deep and prolonged anaesthesia (Dicker, 1953). When a more concentrated solution of ethanol of 20% (v/v) was offered in one of the drinking bottles to eight rats, six of them refused to drink it; the behaviour of these is shown in Fig. 2. Two of eight rats, however, did drink small amounts ( ml./100 g/24 hr) as in Fig. 3. Effect of methylpentynol carbamate on the drinking of ethanol Six rats which had refused to drink 20% (v/v) ethanol were given 12-5 mg methylpentynol carbamate/100 g by mouth for 5 days. Twenty-four hours after the first administration of the drug they began to drink some of the ethanol solution (Fig. 2). The taste for the concentrated solution of ethanol persisted for several days after discontinuance of the drug; all six rats, however, reverted eventually to their initial pattern of drinking water only. As for the two rats which had not shown an initial distaste for a 20 % (v/v) ethanol solution, they refused to drink it as soon as methylpentynol carbamate was administered (Fig. 3). The refusal to drink 20% (v/v) ethanol persisted even after the administration of the drug had been discontinued; and these two rats now refused to drink a 4% (v/v) ethanol solution, which is usually preferred by rats. Irrespective of the effect on ethanol drinking, administration of methylpentynol carbamate (12.5 mg/100 g) decreased the fluid intake in all rats (Figs. 2, 3) without a concurrent decrease of urine excretion.

4 METHYLPENTYNOL IN RATS 141 3~ \ Days Fig. 2. Effect of administration of methylpentynol carbamate on the drinking of a 20% (v/v) ethanol solution; averaged results from 6 rats. -, water drunk (ml./loog/24hr); x -x, urine excretion (ml./100 g/24 hr); *-@, 20% (v/v) ethanol solution drunk (ml./100 g/24 hr); A- - -A, total liquid (water + ethanol solution) intake (ml./100 g/24 hr). Arrows indicate the successive doses of methylpentynol carbamate (12.5 mg/100 g) administered. Note the decrease of fluid intake during the administration of the drug. Effects of methylpentynol carbamate on the water metabolism of rats In an attempt to see whether the unchanged urine excretion during the period of reduced fluid intake was partly due to the diuretic effect of ethanol, a dose of 12-5 mg methylpentynol carbamate/100 g was given for 5 days to control rats allowed free access to food and water. In all six rats, the administration of the drug resulted in a marked degree of hypodipsia; the amount of water drunk fell from to ml./100 g/24 hr without a concurrent decrease of urine excretion, which remained unaffected at ml./100 g/24 h. Food consumption remained at g/100 g/24 hr, but the body weight fell sharply (Fig. 4). Minimizing the progressive dehydration, extrarenal water loss decreased from to ml./100 g/ 24 hr, and the water content of faeces was reduced from 65 to 45 %. After

5 142 S. E. DICKTER the administration of the drug had been discontinued, the volume of water drunk first increased above the normal levels (Fig. 4) before returning to the value before treatment. The normal rate of increase of body weight (0O8 g/100 g/ 24 hr in the present series) was, however, not resumed for at least 7 days after the end of the treatment (Fig. 4) I'' -Ir 12 I I 9I 8 Aj/N -E 48 Fig. 3. I I I I I I I I A, I* * Days Effect of methylpentynol carbamate on rats which drank a 20% (v/v) ethanol solution. The results are the averages from two rats. Symbols as for Fig. 2. Effects of methylpentynol on the release of posterior pituitary hormones It has been shown previously that injections of hypertonic sodium chloride solutions into the carotid artery produce an antidiuretic (Friedman, Hinke & Friedman, 1956) and a pressor effect in the rat (Dicker & Nunn, 1958), and a release of both oxytocic and antidiuretic activities in the dog (Abrahams & Pickford, 1954). The release of these hormones following the intracarotid injection of hypertonic saline solution can be inhibited by ethanol (van Dyke & Ames, 1951; Dicker, 1954). As methylpentynol belongs to the group of alcohols, it was of interest to see whether the observed dissociation between

6 METH YLPENT YNOL IN RATS 143 hypodipsia and urine excretion could be explained by an inhibition of the release of posterior pituitary hormones. Anaesthetized rats, treated with dibenamine, received an injection through the common carotid artery of 0 1, 0-2 or 0 3 ml. of a 3% NaCI solution. This produced rises of blood pressure of the same order as those observed after intravenous (or intracarotid) injections of 1 0, 2-0 or 3 0 m-u. vasopressin, "Vvt % _ ~o1-6 A 6 4 X4 B 3 -/ 2-2~~~~~~~~~~~~~~~ Days EFig. 4. Effects of methylpentynol carbamate on bodly weight and water metabolism of six oontrol rats. 03-&3 water drn (ml./100g/24hr); 0-0, food eaten (g/100g/24 hr); x-x, urine excretion (ml./100 g/24 hr); A---EA, increase in body weight exrpressed as percentage. Arrows indicate daily saiitainof methylpentynol carbamate (12-5 mg/100 g). respectively (Dicker &; Nunn, 1958). After an intracarotid injection of methylpentynol (5 mg/100 g), there was no pressor response to osmotic stimulation though the response to injection of vasopressin remained unchanged (Fig. 5). In anaes9thetized female rats, intracarotid injection of 0 3 ml. of a 3 %/ NaC:l solution produced contraction of the uterus which could be matched by that

7 144 S. E. DICKBR produced by intravenous injections of m-u. oxytocin, as in the example of Fig. 6. After intracarotid injection of methylpentynol (5 mg/100 g) uterine responses to osmotic stimulation of the neurohypophysis disappeared entirely, though the uterus continued to retract normally to further injections of oxytocin. Here and in the earlier experiments of Dicker & NKun (1958) the intracarotid injection of 0'3 ml. of a 3% NaCl solution released from the posterior pituitary gland about times as much oxytocic as pressor activity in the rat, confirming the findings of Abrahams & Pickford (1954) on the bitch; methylpentynol, like ethanol, inhibited the release of both activities. Time, T.m.1.00 P El- C~95F-,g 105: a b a d d Fig. 5. Pressor responses in an anaesthetized rat before and after intracarotid injection of methylpentynol. An increase of blood pressure is shown by a downward line on the tracing. a, response to the intravenous injection of 20 m-u. vasopressin; b, response to the intravenous injection of 1.0 m-u. vasopressin; c, pressor response following the intracarotid injection of 0.1 ml. of a 3% NaCl solution; d, pressor response following the intracarotid injection of 0-2 ml. of a 3% NaCl solution. A dose of 5 mg methylpentynol was injected into the carotid artery at 2 p.m. a b a a b a b a Fig. 6. Contractions of the uterus recorded in 8itu in an anaesthetized rat. a, Intravenous injection of 60 m-u. oxytocin; b, intracarotid injection of 0-3 ml. of a 3% NaCl solution. A dose of 5 mg methylpentynol was injected into the carotid artery 30 min before the beginning of the tracing on the right. Time marker, minutes. DISCUSSION Rats offered both water and weak ethanol solutions showed a marked preference for the latter, which they drank to the practical exclusion of water (Richter, 1957). When the concentration of ethanol was increased above 15% (v/v), preference for ethanol disappeared in the great majority of cases. Two out of eight rats, however, drank small amounts of 20% (v/v) ethanol solution. It is interesting to note that though all the rats used in this investigation were approximately of the same age, these two animals were 90 and 105 g heavier than the average weight of the colony. Richter (1957) has pointed to the possible relation between basal metabolism and 'alcohol craving' in rats.

8 METHYLPENTYNOL IN RATS 145 Little is known about the mode of action of methylpentynol. It is neither an analgesic nor an anaesthetic drug (Margolin, Perlman, Villani & McGavack, 1951); it has, however, a definite hypnotic and sedative effect (Margolin et al. 1951; Halpern, 1956) which, according to Halpern & Lehmann (1956), results from its action on the hypothalamus and on the reticular formation of the brain. Given to rats, it increases both their activity and their exploratory behaviour (Dicker, Steinberg & Watson, 1957) but decreases their ability to learn (Watson, 1958). In men faced with a difficult task methylpentynol appears to impair their performance (Dicker & Steinberg, 1957). From the present investigation it would appear that methylpentynol carbamate can alter the taste of rats for or against ethanol: under the influence of the drug, animals which normally refused to drink 20% (v/v) ethanol solution drink it, and those few rats which drank such concentrated solutions refused to drink ethanol in any concentration. Methylpentynol carbamate has a pronounced effect on the water metabolism of rats: it produces a marked fall of body weight and a noticeable hypodipsia without concurrent decrease of urine excretion. This is reminiscent of the syndrome observed in dogs after electrocoagulation of the lateral part of the anterior hypothalamus (Witt, Keller, Batsel & Lynch, 1952; Andersson & McCann, 1955): following the operation, the dogs lost weight though their food intake remained normal, and they showed marked hypodipsia, though the urine excretion did not appear to be much decreased. Indeed, Witt et al. (1952) suggested the injection of Pituitrin (pituitary extract; Parke, Davis and Co.) as a requisite therapy against the loss of fluid through urine excretion. It is therefore possible that one of the sites of action of methylpentynol lies in the region of the anterior hypothalamus. This hypothesis would agree not only with the 'autonomous depressant effects' of the drug as observed in man (Dicker & Steinberg, 1957) but also with the fact that, like ethanol, it inhibits the release of the neurohypophysial hormones following osmotic stimulation. This may not be surprising if one remembers that methylpentynol is an unsaturated aliphatic carbinol which, like ethanol, belongs to the group of alcohols. Though the mechanism of inhibition is not known, it is possible that both drugs interfere in the hypothalamus with the release of acetylcholine in a way similar to that observed for methylpentynol in a frog nerve-muscle preparation (Nicholls & Quilliam, 1956). If this is so, the difference between their actions would be narrowed to that of a greater potency of methylpentynol. The fact that, in contrast with ethanol, methylpentynol is not attacked by alcohol-dehydrogenase (A. L. Greenbaum, personal communication) may account for this difference. 10 PHYSIO. CX2LIV

9 146 S. E. DICKER SUMMARY 1. Rats given a choice of water or ethanol solutions to drink showed a clear preference for the latter, as long as the concentration of ethanol did not exceed 15% (v/v). 2. When both a 20% (v/v) ethanol solution and water were offered, the majority of rats drank water only. Under the influence of methylpentynol carbamate, however, they drank some of the 20% (v/v) ethanol solution. 3. Administration of methylpentynol carbamate (12-5 mg/100 g/day for 5 days) to control rats kept in metabolism cages with free access to food and water produced a marked hypodipsia without concurrent decrease of urine flow. The food intake remained unchanged, but the body weight decreased appreciably. 4. Intracarotid injection of 5 mg methylpentynol in anaesthetized rats prevented the release of both pressor and oxytocic activities in response to osmotic stimulation of the neurohypophysis. It is suggested that one of the possible sites of action of methylpentynol lies in the anterior part of the hypothalamus. I wish to thank Miss Joan Nunn for her technical skill and her constant help. REFERENCES ABRAHAMS, V. C. & PICKFORD, M. (1954). Simultaneous observations on tht, rate of urine flow and spontaneous uterine movements in the dog, and their relationship to posterior lobe activity. J. Phy8iol. 126, AwDERssN, B. & McCANN, S. M. (1955). The effect of hypothalamic lesions on the water intake of the dog. Acta phy8iol. 8cand. 35, CusHwy, A. R. (1897). On the effects ofelectrical stimulation of the mammalian heart. J. Phy8o. 21, DEKANSKi, J. (1952). The quantitative assay of vasopressin. Brit. J. Pharnucol. 7, DICKER, S. E. (1953). A method for the assay of very small amounts of antidiuretic activity with a note on the antidiuretic titre of rats' blood. J. Physiol. 122, DIcKER, S. E. (1954). The fate of the antidiuretic activity of pitressin in rats. J. Phy8iol. 124, DICKER, S. E. (1958). The effect of methylpentynol on the water metabolism of rats. J. Physiol. 142, 29P. DICmER, S. E. & NuXNN, J. (1957). The role of the antidiuretic hormone during water deprivation in rats. J. Physiol. 136, DICKER, S. E. & NUNN, J. (1958). Antidiuresis in adult and old rats. J. Phy8iol. 141, DICKER, S. E. & STEINBERG, H. (1957). The effect of methylpentynol in man. Brit. J. Pharmacol. 12, DIcBm, S. E., STEINBERG, H. & WATSON, R. H. J. (1957). Effect of methylpentynol on the activity of rats. J. Physiol. 137, 88-89P. EGGLETON, G. (1942). The diuretic action of alcohol in man. J. Physiol. 101, FREDMAN, S. M., HINKE, J. A. M. & FRIEDMAN, C. L. (1956). Neurohypophyseal responsiveness in the normal and senescent rat. J. Geront. 11, GTiLLSPIE, R. J. G. & LuCAS, C. C. (1958). An unexpected factor affecting the alcohol intake of rats Canad. J. Biochem. Physiol. 36, HALPTEN, B. N. (1956). Propri6t6s sedatives et anticonvulsivantes du carbamate de methyl-3- pentyne-1-ol-3. C.R. Soc. Biol., Pari8, 150,

10 METHYLPENTYNOL IN RATS 147 HRzium, B. N. & LEHrw, A. (1956). Action protectrice du carbamate de m6thyl-3-pentyne- 1-ol-3 contre la crise audiogone. C.R. Soc. Biol., Paris, 150, HATPERN, B. N. & LEwisr, A. (1957). Bases exp6rimentales de I'action th6rapeutique d'une nouvelle m6dication s6dative et antianxieuse, le carbamate de methyl-3-pentyne-1-ol-3. Pr. md. 65, OLINi, S., PBRXM.&.1, P., VIu&m, F. & McGAVACK, T. M. (1951). A new class of hypnotics: unsaturated carbinols. Science, 114, NIcHOELS, J. G. & QuuM, J. P. (1956). The mechanism of action of paraldehyde and methylpentynol on neuromuscular transmission in the frog. Brit. J. Pharmacol. 11, RiCHTo=, C. P. (1957). Production and control of alcoholic cravings in rats. In Abramson, H.A., Neuropharmacology. New York: Josia Macy, Jr. Foundation. va. DYKc, H. B. & AxEs, R. G. (1951). Alcohol diuresis. Acta endocr., Copenhagen, 7, WATSON, R. H. J. (1958). Effect of methylpentynol on learning in rats. J. Physiol. 142, 30P. Wnr, D. M., KzuLsu, A. D., BATSEL, H. L. & LYNCH, J. R. (1952). Absence ofthirst and resultant syndrome associated with anterior hypothalamectomy in the dog. Amer. J. Phy8iol. 171, 780P. 10-2